Team:Freiburg/Protocols/Purification
Protein Purification on gravity flow columns
improved protocol for classical gravity flow columns
material:Gravity flow columns, 50% Ni-NTA agarose in storage buffer, ddH<sub<2</sub>O, Lysis buffer
duration:
All buffers have to be degassed before by applying sonfication for example
To increase the flow though velocity the collection tubes (falcon tubes with cap) were connected to a vacuum pump with interposed pressure regulation. beware of applying too much pull to avoid ait bubble formation near the diaphragma.
column packing
- wash columns with 5 mL of aqua dest
- close columns with plug
- slowly pipette 1 mL of 1:1 NTA-agarose/stoarge buffer slurry on the diaphragma
- let the agarose settle out
- open the tube and wash the agarose with 2-3 mL lysis buffer
loading
- change the falcon tube to elute the sample in
- pipette the cell lysate onto the column and let almost flow through
- collect flow-through and change collection tube again
- wash with 5 mL washing buffer and collect FT
- change collection tube
- elute 5 times with 500 µL of elution buffer
- every time the liquid surface is near the bed: add new buffer and collect the flow though that accumulated until then
washing
- add 500µL - 1mL of total elution buffer and let flow through
- wash with 3 mL of aqua dest to remove imidazol
- add 1 mL of 20 % ethanol and let flow though until around 0.5 mL left
- close column with plug and store @ 4°C
reusing
- equilibrate the column with lysis buffer as described above
- use one column only for one type of protein!
| buffers | |
|---|---|
| standard buffer/lysis buffer | 20 mM Tris pH 7.5 |
| 150 mM NaCl | |
| 0.3 mM PMSF | |
| 10mM Imidazole | |
| washing buffer | 20 mM Tris pH 7.5 |
| 150 mM NaCl | |
| 20 mM Imidazole | |
| elution buffer | 20 mM Tris pH 7.5 |
| 150 mM NaCl | |
| 500 mM Imidazole | |
